The objectives of the proposed research program are to elucidate the fundamental mechanisms of RNA splicing and the relationship of abnormal RNA splicing to deficient gene expression. Intervening sequences are present in a number of eukaryotic genes and viruses; however, the function of these sequences, and the means of their removal from the nuclear precursor RNA is not known. I will examine the structural, temporal, and biochemical parameters of the RNA splicing process using the human globin genes as a model system, and will continue my investigation of the role of abnormal RNA splicing in the pathogenesis of the disorder. I shall also investigate the utility of a potential means for the prenatal diagnosis of this disorder. In order to investigate the splicing of globin precursor RNAs, segments of previously cloned normal and thalassemic human globin genes which contain intervening sequences will be cloned in SV40 virus, and the processing of the resultant hybrid RNAs in infected cells studied by gel blotting and rapid RNA sequencing methods. A second line of investigation will begin to identify and characterize those nuclear proteins which are involved in the RNA splicing reaction itself. In order to identify these proteins, I will utilize the "protein blotting" technique to identify RNA-binding proteins which specifically recognize the intervening sequences in precursor RNAs. If such proteins can be identified, purification will be approached utilizing an affinity chromatography method, using purified intervening sequence RNA segments to bind the specific proteins. The ultimate goal of this line of investigation is the characterization of the proteins invovled in RNA splicing, and of the manner in which they interact with RNA. I shall also investigate the utility of a polymorphism of Msp I cleavage which I have identified which is associated with the Beta+-thalassemia gene. In collaboration with Dr. Bernard Forget at Yale Univeristy, DNAs from normals and thalassemic patients will be screened in order to establish the frequency of this polymorphism in the normal and thalassemic populations.